This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Living cells interact with and respond to their environment through networks of proteins that transduce extracellular signals into specific biochemical responses inside the cell. Specificity within each pathway is essential for proper regulation of diverse cellular behaviors. Scaffold proteins have been suggested to contribute to specificity by tethering individual signaling proteins together into multi-protein complexes. Emerging evidence suggests that scaffold proteins can also play a more direct role in signal transmission by allosterically promoting specific kinase-kinase reactions and by undergoing conformational changes in response to upstream activators. To construct a physical model for how scaffold proteins promote specific kinase-kinase reactions, we will use a combination of kinetic and biophysical approaches. First, we will measure rate constants for individual kinase-kinase reactions in the presence and absence of scaffolds. Second, we will identify key surfaces and interactions required for scaffold action using crystallographic and biochemical methods. A necessary component of this project is the ability to determine if different protein samples have been phosphorylated, and to identify specific sites of phosphorylation in target proteins. The UCSF mass spectrometry facility will be instrumental in obtaining this information. These experiments will provide kinetic and thermodynamic framework for scaffold-mediated signal transduction, which will provide the basis for understanding how specificity is maintained in cell signaling.